In order to improve the notoriously poor outcome of patients with malignant glioma, we developed a novel oncolytic adenovirus, Delta-24-RGD. In the initial funding period of this SPORE grant, we made the significant translational step of completing a first-in-human phase I clinical trial in patients with recurrent malignant glioma (NCT00805376). In this trial several dramatic complete responses were seen, and analyses of post treatment surgical specimens proved for the first time that Delta-24-RGD was capable of replicating in and killing human tumor cells, emphasizing the urgent need to further develop this approach. However, our analyses also suggested that the efficacy of Delta-24-RGD could be improved by 1) augmenting viral spread, and 2) improving the method of delivery. In this context, preclinical studies proposed in Aim 2 of our initial grant showed that the efficacy of Delta-24-RGD was synergistically enhanced by combining it with temozolornide (TMZ). Other observations showed for the first time that viral mediated autophagy and autophagy-related cell death are critical to oncolysis, and that promoting autophagy may further improve the efficacy of Delta-24-RGD. Equally important, preclinical studies from Aim 3 of our initial proposal showed that the delivery of Delta-24-RGD could be improved by the use of intravascularly administered bone marrow mesenchymal stem cells (BM-hMSCs) loaded with Delta-24- RGD. Based on these results, we now hypothesize that the efficacy of Delta-24-RGD can be enhanced without adversely affecting safety by combining Delta-24-RGD with TMZ, by harnessing autophagy, and by improving delivery via BM-hMSCs. To test this hypothesis, we wi|l: explore the role of autophagy in the synergy of TMZ and viral oncolysis using glioma stem cells, and develop a next generation autophagy- targeted oncolytic adenovirus (Aim 1); assess the safety, efficacy, and biological effects of combining Delta-24-RGD with TMZ in a phase l/ll clinical trial (Aim 2), and validate the effectiveness of BM-hMSCs to delivery Delta-24-RGD in preclinical glioma stem cell models and in patients with recurrent GBM. This project is the next step in achieving our long-term goal of legitimizing these viral and stem cell therapies as standard treatments of malignant gliomas. RELEVANCE: DO NOT EXCEED THE SPACE PROVIDED. Because malignant gliomas are among the most deadly human cancers, enhancing Delta-24-RGD anti- glioma effects represents a major step forward in broadening our arsenal of treatments for patients with this deadly disease for whom there are currently only minimally effective therapies